Experiments are proposed here to investigate the molecular and cellular nature of the DNA protein crosslinks generated by hyperthermia and radiation. The hypothesis is that the thermal enhancement of radiation induced DNA protein crosslinks results from the known increased associated of protein to DNA by hyperthermia. There is, by protein availability, an increase in radiation induced DNA protein crosslinking. Further, these generated crosslinks are in part responsible for the lethality of heat and radiation through the crippling of chromatin replication and repair. First, at the cellular level experiments are designed around the following points: a) Conditions will be defined to maximize the frequency of DNA protein crosslinks, since one would expect the conditions of maximal lethality and DNA-protein crosslinking to correspond. These experiments will test the link between DNA protein crosslinks and lethality, and will utilize the known dependence of thermal radiosensitization on the cell cycle. b) If protein availability is a factor, the known thermal conditions for maximizing nuclear proteins should result in maximizing the DNA-protein crosslinks. In addition, since the thermal enhancement of nuclear protein is independent of cellular metabolism, the increase in DNA protein crosslinks should also be non-metabolic in nature. c) Cellular factors effecting the removal of DNA-protein crosslinking. d) The extent DNA protein crosslinking should correlate with defective DNA metabolism. For example, if DNA protein crosslinking primarily occurs on replicons, one would expect a dependence of DNA protein crosslinking on the frequency of replicon initiation (namely, the position of a cell in S phase). Second, at the molecular level, the nature of the DNA protein crosslinks known to occur will be scrutinized to discover a) whether a covalent bond exists between the nucleotides and the proteins and if so which protein residue is involved and b) the proteins and the DNA fragment which are crosslinked. Biochemically, both the DNA sequence and the proteins can be characterized. These experiments are feasible and potentially important if the correlative evidence implicates these DNA protein crosslinks with the processes of DNA replication, repair and cell viability.